Abstract DNA nanoparticles have been tested in CF patients and shown to be safe and able to deliver nucleic acids to the airway. We have reported that nucleolin interaction with glucocorticoid receptor (GR) is essential for cellular binding and transfection of DNPs and discovered that DNPs interact with other proteins in human primary airway epithelial cells, including adenomatous polyposis coli (APC), and non-erythrocytic spectrin alpha 1 (SPTAN1). These interactions can significantly modulate DNPs-mediated gene transfer, which prompts our hypothesis that DNP interaction with GR, HSP90, APC, and SPTAN1 modulate nucleolin-mediated transport of the particles and that the interactions can be manipulated with agents approved in humans to enhance gene delivery in vivo. To investigate the hypothesis, our specific aims are: 1) To evaluate the impact of manipulating the DNP interactome on gene delivery and toxicity in vivo; and 2) To examine the mechanisms that enhance gene transfer in well differentiated airway epithelial cells from CF patients. The proposed studies will be the first to explore the biology of DNPs and improve our understanding of important steps in delivery. Furthermore, the studies have the potential of defining small molecules already approved for use in humans as agents that can be administered before or during nanoparticle administration to significantly enhance gene transfer of CFTR into the therapeutic range.